Acquired Immunodeficiency Syndrome (AIDS) is characterized by immunosuppression that results in opportunistic infections and malignancies; wasting syndromes; and central nervous system degeneration. Destruction of CD4+ T-cells, which are critical to immune defense, is a major cause of the progressive immune dysfunction that is the hallmark of AIDS disease progression. The loss of CD4+ T cells seriously impairs the body's ability to fight most pathogens, but it has a particularly severe impact on the defenses against viruses, fungi, parasites and certain bacteria, including mycobacteria.
AIDS is caused by infection with human immunodeficiency virus (HIV). An infectious HIV particle includes two strands of RNA packaged within a viral protein core. The core is surrounded by a phospholipid bilayer envelope derived from a host cell membrane that also includes virally-encoded membrane proteins.
The HIV genome encodes several structural proteins and has the characteristic 5′-LTR-Gag-Pol-Env-LTR-3′ organization of the retrovirus family. The env gene encodes the viral envelope glycoprotein (Env) that is translated as a 160-kilodalton (kDa) precursor (gp160) and cleaved by a cellular protease to yield an external 120-kDa envelope glycoprotein (gp120) and a transmembrane 41-kDa envelope glycoprotein (gp41). These glycoproteins are required for HIV to infect cells.
HIV infection begins when gp120 on the viral particle binds to CD4 and chemokine receptors on the cell membrane of a subject's target immune system cells (e.g., CD4+ T-cells, macrophages and dendritic cells). The bound virus then fuses with the target cell and reverse transcribes its RNA genome. The resulting viral DNA integrates into the subject cell's genome and begins to produce new viral RNA, resulting in new viral proteins and virions. The virions leave the originally infected cell to then infect new cells. This process kills the originally infected cell.
HIV-1 broadly neutralizing antibodies (bNAbs) are antibodies capable of neutralizing HIV. HIV bNAbs target four major areas of the Env: (i) the membrane proximal external region of the gp41 subunit; (ii) the CD4 receptor-binding site (CD4-BS); (iii) two sites including both carbohydrate and amino acid moieties, one at the base of the “V3” and another on the “V1/V2” loops of the gp120 subunit; and (iv) regions spanning elements of both gp120 and gp41.
Based on their ontogenies and mode of recognition, the CD4-BS bNAbs are grouped into two major types: (i) heavy chain complementary determining region three (CDRH3)-dominated; and (ii) variable heavy (VH)-gene-restricted. Antibodies that make contact primarily through their CDRH3 regions are further subdivided into the CH103, HJ16, VRC13 and VRC16 classes while the VH-gene-restricted Abs include the VRC01- and the 8ANC131-class antibodies.
VRC01-class bNAbs protect non-human primates from experimental simian/human HIV (SHIV)-infection and humanized mice from HIV-1 infection. It was therefore thought that vaccine-elicited VRC01-class bNAbs would protect humans from HIV-1 infection. With the exception of llamas however, all efforts to elicit such antibodies by immunization in humans or wild type animals with recombinant Env (rEnv) have been unsuccessful.
One of the many important reasons for the lack of success is thought to be the inability of the Env proteins used as immunogens to engage B cell receptors (BCRs) that encode the germline (gl) of VRC01-class antibodies (e.g., “immature” or not fully developed Abs). Indeed maturation of these antibodies to full neutralizing Abs requires that they circumvent steric constraints on Env through extensive somatic hypermutation. For example, HIV-1 has evolved to avoid detection by gl B cells that give rise to VRC01-class bNAbs through development of specific N-linked glycosylation sites (NLGS) (for example, in Loop D and V5 of the gp120 subunit). As a consequence, recombinant Env proteins derived from diverse HIV-1 isolates are ineffective in binding to and stimulating B cells engineered to express the glBCR forms of VRC01-class bNAbs in vitro. Targeted disruption of conserved NLGS at position 276 in Loop D, and at positions 460 and 463 in V5 of the 426c clade C Env, however, permits binding and activation gl B cell lines expressing BCRS of two clonally-related VRC01-class bNAbs, VRC01 and NIH45-46 in vitro. These two BCRs represent a small subset of potential VRC01-class antibody progenitors. Thus, designing immunogens capable of recognizing a larger group of glVRC01-class BCRs should increase the chances of activating rare, naïve glVRC01-class B cells during human immunization.
Previous reports describe preparation of artificial gp120 proteins (in some instances called “engineered outer domain” or “eOD” proteins) for use as HIV vaccines. The previous reports also describe multimerizing the artificial gp120 proteins to enhance immunogenicity of the proteins, and more particularly, the VRC01 epitope of the proteins. It was envisioned that multimerized artificial gp120 proteins would stimulate gl B cells using multivalent avidity, and, further, that the addition of the larger particulate forms would mimic a virus-like symmetric presentation of epitopes, reduce immune responses to regions buried in the multimer, and enhance in vivo trafficking of the artificial gp120 proteins to lymph nodes.
These previous reports described eOD proteins that lack normally occurring loops. The eOD proteins were also multimerized as trimers, tetramers, and octamers using coiled-coil multimerization domains. From the trimers and tetramers, octamers, 24mers, 60mers, and 180mers were formed.